Selection of tunicamycin-resistant Chinese hamster ovary cells with increased N-acetylglucosaminyltransferase activity.

Abstract

Chinese hamster ovary (CHO) cells resistant to the antibiotic tunicamycin (TM) have been isolated by a stepwise selection procedure with progressive increments of TM added to the medium. TM inhibits asparagine-linked glycoprotein biosynthesis by blocking the transfer of N-acetylglucosamine-1-phosphate from UDP-N-acetylglucosamine to the lipid carrier. The TM-resistant cells exhibited a 200-fold increase in their LD50 for TM and were morphologically distinct from the parental cells. The rate of asparagine-linked glycoprotein biosynthesis was the same for wild-type and TM-resistant cells. Membrane preparations from TM-resistant cells cultured for 16 d in the absence of TM had a 15-fold increase in the specific activity of the UDP-N-acetylglucosamine:dolichol phosphate N-acetylglucosamine-1-phosphate transferase as compared to membranes of wild-type cells. The products of the in vitro assay were N-acetylglucosaminylpyrophosphoryl-lipid and N,N'-diacetylchitobiosylpyrophosphoryl-lipid for membranes from both TM-resistant and wild-type cells. The transferase activity present in membrane preparations from wild-type of TM-resistant cells was inhibited by comparable levels of TM. The data presented are consistent with overproduction of enzyme as the mechanism of resistance in these variant CHO cells.